It is a primary goal of research efforts in plant biotechnology to genetically engineer plants so that they have a new or improved trait or characteristic. A specific example of great commercial interest is the production of commercially useful recombinant proteins in plants by a process known generally as plant molecular farming, (U.S. Pat. Nos. 4,956,282 and 5,550,036, Fischer et al., 2004, Spök et al., 2007).
Seeds are particularly useful as vehicles for the production of proteins for molecular farming applications as they are naturally developed for innate storing ability and stability. Examples of plants that have been engineered for seed specific expression and could be used for these purposes include corn, wheat, flax, sunflower, rice, soybean, canola, peanut, tobacco, Arabidopsis and castor, (U.S. Pat. Nos. 5,650,554, 5,693,506, and 7,157,629).
Examples of the types of transgenic molecular farming peptide or protein products that have been made in seed include enzymes, (U.S. Pat. Nos. 5,804,694, 6,087,558, 6,800,792, 5,994,628 and 7,390,936), enzyme inhibitors, (U.S. Pat. No. 5,824,870), structural proteins, (U.S. Pat. No. 6,617,431), hormones and growth factors, (U.S. Pat. Nos. 6,288,304 and 7,091,401), antibodies, (U.S. Pat. No. 6,417,429) and vaccine antigens, (U.S. Pat. Nos. 5,914,123, 5,654,184, and 6,761,914).
Although a wide range of different recombinant proteins have been made, and in some instances purified from seed, many of these processes have been undertaken using seeds of plants that constitute major food crops such as corn, rice, barley and canola. The use of major food crops for molecular farming purposes has caused regulatory and public acceptance concerns because of possible contamination of the food supply with unwanted recombinant products. The possibility of gene flow from these plants to wild relatives has also been cited as an environmental issue, (Hills, M., et al., 2007). Molecular methods to impede or block transgene movement have been developed, (Schernthaner, J. P., et al., 2003, U.S. Pat. Nos. 5,977,441, 5,723,765, 5,925,808, 7,452,986, 7,671,253, 8,124,843) but have not been widely used for a variety of reasons that are not related to the functionality of the technology.
Commonly used and easily transformed non-food species such as tobacco or Arabidopsis could be contemplated for molecular farming but these species have small seeds which makes larger scale production impractical. The large-seeded non-food crop Castor is problematic due to the severe toxicity of this species.
Tarwi, (Lupinus mutabilis Sweet) is a large-seeded legume that has been known and used since ancient times in South American Andean countries but is not grown in North America. It is adaptable, semi-hardy, nitrogen fixing and the mature plant, frost tolerant. Tarwi seeds contain an average of 46% protein, have a high lysine content, and contain 20% lipid, providing a nutritional balance equivalent to soybean. Some varieties have a high content of water soluble alkaloids that are easily removed by washing seed that has been soaked.
Large-seeded legumes are generally recalcitrant to transformation. A method of transforming beans is disclosed in WO 2012/0121494. However, it is known in the art that methods for transforming one type of legume typically do not translate well to other types of legumes. The type of tissue that is to be transformed and the various media combinations required are unpredictable and need to be developed specifically for individual species and even cultivars.
Tissue culture investigations of L. mutabilis have been limited but include: micropropagation (Pniewski et al. 2002), regeneration from thin-cell layers (Mulin and Bellio-Spataru, 2000) and somatic embryogenesis from immature cotyledons (Nadolska-Orczyk 1992). Genetic transformation has been reported for Lupinus species such as L. angustifolius L. (Pigeaire et al. 1997; Molvig et al. 1997) and L. luteus (Li et al. 2000).
There has been only one report of successful genetic transformation of L. mutabilis (Babaoglu et al. 2000). This study used shoot apical explants as the transformation target which is time consuming and impractical for large scale transformation studies (Babaoglu et al., 2000).
The process of expressing a desired gene in a plant involves constructing a vector that comprises the gene of interest downstream of a promoter, introducing the vector into a plant, and expressing the gene in the plant. In situations where stable expression of the gene is desired, the gene is incorporated into the genome. Generally, the foreign DNA is integrated into nuclear DNA, however methods for the integration of foreign DNA into the plastid genome have been developed for some plant species (U.S. Pat. Nos. 5,451,513; and 5,693,507).
It is desirable to be able to direct gene expression to a specific organ, such as a seed, in order to facilitate harvesting of proteins and to avoid protein production in other tissues which may have adverse effects on plant health or plant growth or could raise regulatory concerns. Researchers have identified a number of promoters that are useful for the expression of genes in seed. These include promoters related to storage gene products, seed components such as embryo, endosperm, ovule, seed coat etc., promoters active in early or late development and those that are specific to individual species. In legume seed described promoters include: a soybean lectin promoter; the conglycin promoter; the widely used 7S b-phaseolin storage protein promoter (U.S. Pat. No. 5,504,200); and the LegA2 Pisum sativum legumin gene promoter.
The LegA2 gene promoter from pea is described in Lycett Q. W., et al., (1985). This promoter was shown to function in tobacco, (Ellis et al., 1988). The legumin seed promoter has also been described from pigeon pea, (Jaiswal, R., et al., 2007), and more recently from non-leguminous species sorghum and maize (U.S. Pat. Nos. 7,897,841, 7,622,637 and 7,211,712).
Many different procedures have been described that physically introduce foreign DNA into plant cells. A common strategy has been the “biolistic” acceleration of small dense carrier particles, such as particles of gold that are coated in foreign DNA, by what is known in the art as a “gene gun” (U.S. Pat. No. 4,945,050; 5,036,006; and 5,371,015). A variety of different “gene guns” for shooting DNA into plant cells have been developed (Ziolkowski, 2007; U.S. Pat. Nos. 5,976,880; 5,584,807). Other physically carriers such as tungsten “whiskers” or silicon carbide crystals have also been used to deliver foreign DNA by puncture of the cell wall creating channels for DNA entry (Asad, S., et al. 2008; U.S. Pat. Nos. 5,302,523, 5,464,765; 7,259,016: 6,350,611).
Other physical approaches have included the micro-injection of DNA solutions directly into cell nuclei, (U.S. Pat. Nos. 4,743,548, 5,994,624) and production of pores in cellular membranes for DNA uptake with electric currents (U.S. Pat. No. 6,022,316). The removal of the external cell wall barrier and preparation of protoplasts facilitates the uptake of DNA directly from solution but in some instances regeneration of plants from protoplasts is challenging (U.S. Pat. Nos. 4,684,611; and 5,453,367).
By far the most widely practiced general method of achieving plant transformation has been by the use of disarmed strains of Agrobacteria (as reviewed in Gelvin, 2003). Agrobacterium tumefaciens and related soil bacteria naturally comprise a DNA plasmid (i.e. a T-DNA plasmid) that is physically mobilized into plant cells by bacteria proliferating in a wound site. The T-DNA plasmid has left and right border sequences that are required for integration of DNA into the plant host genome. Foreign DNA between the border sequences is thus selectively introduced into the host genome.
Naturally occurring Agrobacterium species introduce foreign DNA that comprises genes for the production of plant growth regulatory substances and uncommon amino acid metabolites known as opines. This results in the formation of a tumour at the site of infection that in addition to providing a refuge for the growth of Agrobacteria supplies specific nutrients beneficial to the bacteria. The formation of crown gall tumours, (or hairy root proliferation) by Agrobacterium sp. is an example of molecular parasitism. Naturally occurring plasmids have been modified, “disarmed” by removal of genes that cause tumor formation and support bacterial growth. The Ti plasmid was also modified to remove so-called virulence factors needed for DNA transfer. These factors were placed on a separate plasmid so that only selective recombinant DNA is added to the host plant cells and not the Vir genes. The technique of removal of the virulence factor DNA to a separate plasmid is known as “disarming” and resulted in the development of the preferred binary transformation method (U.S. Pat. No. 4,940,838).
Initially, it was felt that Agrobacterium mediated transformation only occurred with dicot species however over time Agrobacterium strains that infect monocots were discovered and transformation using Agrobacterium was demonstrated (U.S. Pat. Nos. 5,591,616; and 7,060,876).
An important consideration for regeneration of transformed plants is the tissue targeted for biolistic or Agrobacterium mediated transformation. Tissue targets that have been shown to be useful for the regeneration of transformed plants include: leaf discs, stem segments, petioles, decapitated meristems, roots, flower buds and pollen. Any tissue can be used that can subsequently be regenerated into whole functional transgenic plants.
An example of a therapeutically useful enzyme is adenosine deaminase (E. C. 3.5.4.4). In humans, adenosine deaminase is needed for the breakdown of adenosine from food and the turnover of nucleic acids in tissues. A primary function of this enzyme is in the development and maintenance of the immune system. The ADA active site contains a zinc atom, the only cofactor needed for enzymatic activity.
Mutations in the adenosine deaminase gene result in reduced or a complete lack of expression resulting in a disease condition known as Severe Combined Immune Deficiency, (SCID). SCID is considered an orphan disease, occurring with a frequency of less than one in 100,000 live births worldwide. ADA is needed to break down metabolic byproducts that become toxic to T-cell lymphocytes. Most other cells have alternate means of removing these byproducts and are less affected by ADA deficiency. T-cells of SCID individuals die a few days after being produced in contrast to a normal life span of a few months. Consequently, T-cell numbers are greatly depleted. Because T-cells control B-cell activity, the reduction in T-cells results in the absence of both T-cell and B-cell function resulting in severe combined immune deficiency. SCID victims are unable to mount an effective immune response to any infections, a defect that is soon fatal.
Several clinical approaches for SCID have been explored including bone marrow transplants and gene replacement therapy, however the most widely used approach is via enzyme replacement therapy. Patients receive weekly or twice weekly injections of the ADA enzyme which is typically presented in a PEGylated form to slow degradation. The ADA used for replacement therapy is derived from bovine sources. Currently, the cost to supply SCID patients with bovine ADA is in excess of 100,000 dollars per annum per patient. ADA may have additional therapeutic uses in treatment of other immune disorders, (U.S. Pat. No. 5,728,560), HIV (Martinez-Navio et al., 2011) and cancer, (U.S. Patent application 20090047270).
The human form of the enzyme is inherently less stable than the bovine enzyme due to a reactive unpaired cysteine on the enzyme surface. The bovine enzyme has the same number of primary cysteine residues in the same locations as the human enzyme but the reactive cysteine is post-transcriptionally capped with an additional cysteine residue. The primary amino acid sequence of human ADA has been known for some time (Daddona et al., 1984) which has allowed researchers to design modifications and amino acid substitutions to reduce instability (U.S. Pat. No. 8,071,741).
Although it is now known how to increase the stability of recombinant ADA, the human enzyme is largely available in a crude form made in E. coli. Filpula et al, (U.S. Pat. No. 8,071,741) exemplify the substitution in hADA of the reactive cysteine with serine but only contemplate recombinant production in bacteria and yeast.
Transient transformation of tobacco BY-2 cells with hADA genes for the purpose of producing biologically active hADA has been proposed (Singhabahu et al., 2010; Singhabahu and Bringloe 2012). It appears that the researchers were able to isolate ADA from cell cultures of transgenic tobacco cells and transgenic calli, but it was not demonstrated that the isolated protein is active or stable. The use of transient systems for molecular farming has the disadvantage that a desired product must be harvested or isolated immediately.
Thus there remains a need for methods of safely producing large amounts of therapeutically useful enzymes, such as hADA. In addition, there remains a need for efficient and reproducible methods for transforming Lupinus mutabilis. 